Parvovirus capsids

ABSTRACT

The present invention relates to a method of producing non-infections parvovirus capsids and to diagnostic assays and vaccines utilizing same. The invention further relates to recombinant baculoviruses encoding parvovirus structural proteins and host cells infected therewith. The invention also relates to a method of packaging and delivering genetic information utilizing the noninfectious capsids.

This application is a continuation of application Ser. No. 08/407,939filed Mar. 21, 1995, now U.S. Pat. No. 6,132,732, which is a division ofapplication Ser. No. 07/612,672 filed Nov. 14, 1990, now U.S. Pat. No.5,508,186, which is a continuation-in-part of application Ser. No.07/270,098 filed on Nov. 14, 1988, now abandoned, which are herebyincorporated in their entirety by reference.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates, in general, to a method of producingparvovirus antigens, and in particular, to a method of producing empty,and thus non-infectious, parvovirus capsids, and to diagnostic assaysand vaccines utilizing same. The invention also relates to a method ofpackaging and delivering genetic information using the empty parvoviruscapsids. The invention further relates to a method of packing anddelivering nonparvovirus proteins, such as other antigens, ligands andenzymes, using empty parvovirus capsids.

2. Background Information

Parvoviruses are common agents of animal disease. The first strong linkbetween parvovirus infection and human disease came from theserendipitous discovery in 1975 of parvovirus-like particles in the seraof normal human blood donors (one of the samples having been designatedB19). Since that time, B19 parvovirus has been identified as thecausative agent of: i) transient aplastic crisis (TAC) of hemolyticdisease, ii) the common childhood exanthem called fifth disease; iii) apolyarthralgia syndrome in normal adults that may be chronic andresembles in its clinical features, rheumatoid arthritis; iv) some casesof chronic anemia and/or neutropenia; and v) some cases of hydropsfetalis. The entire spectrum of human illness caused by parvoviruses,however, is not yet clear due, in large part, to the fact that anappropriate assay is not widely available.

Parvoviruses require replicating cells for propagation, and parvovirusinfection, therefore, results in pathologic changes in mitoticallyactive host tissue. In infected children and adults, B19 parvovirusreplicates in the bone marrow; in the fetus, B19 parvovirus replicatesin the liver, there a hematopoietic organ. Erythroid progenitor cellsare the only cell type known to be subject to infection by this virus.

The limited host and tissue range of B19 parvovirus has hampered thedevelopment of assays specific for the virus. Since the discovery of thevirus, the quantity of B19 antigen available as a reagent has beenlimited to that obtainable from sera fortuitously obtained from infectedpatients. The virus has an extraordinary tropism for human erythroidprogenitor cells and has only been propagated in human bone marrow cellcultures (Ozawa et al. Science 233:883 (1986)), fetal liver (Yaegashi etal. J. Virol. 63:2422 (1989)) and, to a much lesser degree, inerythroleukemia cells (Takahashi et al. J. Inf. Dis. 160:548 (1989)).The bone marrow cultures, however, require explanted bone marrow cellsand, therefore, are not practical for virus propagation. The developmentof and availability of clinical assays continue to be limited by theavailability of the antigen. The production of stable transformantscapable of producing B19 protein products has been prevented by the factthat some of these products are lethal to transfected cells.

SUMMARY OF THE INVENTION

It is a general object of the invention to provide a method of producinglarge quantities of parvovirus antigens.

It is a specific object of the invention to provide a method ofeffecting the expression of parvovirus structural proteins in cellculture.

It is another object of the invention to provide non-infectiousparvovirus capsids.

It is a further object of the invention to provide a safe and effectivemethod of producing antibodies against parvovirus capsid proteins.

It is a still further object of the invention to provide a vaccineeffective against parvovirus infection.

It is another object of that invention to provide diagnostic assays fordetecting the presence in biological samples of parvovirus particles orantibodies thereto.

It is a further object of the invention to provide a method of treatinghemoglobinopathies, enzyme deficiency states and other diseases that maybe amenable to genetic therapy.

It is another object of the present invention to provide a method ofpresenting antigens, ligands and enzymes utilizing the parvoviruscapsids.

Further objects will be clear to one skilled in the art from thefollowing detailed description of the present invention.

In one embodiment, the present invention relates to a method ofproducing parvovirus capsids comprising the steps of:

i) introducing into a host cell a recombinant DNA molecule comprising:

a) an expression vector, and

b) a DNA sequence encoding the structural proteins of a parvovirus, withthe proviso that genes encoding non-structural parvovirus protein arenot included in the DNA sequence;

ii) culturing the cells under conditions such that the structuralproteins are produced and self assemble to form the capsids; and

iii) isolating the capsids.

In another embodiment, the present invention relates to a parvovirusantigen consisting essentially of a parvovirus capsid.

In a further embodiment, the present invention relates to a parvovirusantigen consisting essentially of a parvovirus capsid of majorstructural proteins free of minor structural proteins.

In yet another embodiment, the present invention relates to a diagnosticassay for parvovirus infection comprising:

i) contacting a sample from a patient suspected of being infected withparvovirus with the above-described parvovirus capsid, and

ii) detecting the formation of a complex between anti-parvovirusantibodies present in the sample and the parvovirus capsid.

In another embodiment, the present invention relates to ananti-parvovirus vaccine comprising the above-described parvovirus capsidand a pharmaceutically acceptable carrier.

In another embodiment, the invention relates to a method of packagingand transferring genetic information comprising

i) encapsidating the genetic information in the above-describedparvovirus capsid and

ii) introducing the encapsidated information into a host cell.

In yet another embodiment, the present invention relates to a diagnostickit comprising:

i) the above-described parvovirus capsid; and

ii) ancillary reagents.

In a further embodiment, the present invention relates to a recombinantbaculovirus comprising a DNA segment encoding a minor structural proteinof a parvovirus and to a recombinant baculovirus comprising a DNAsegment encoding a major structural protein of a parvovirus.

In another embodiment, the present invention relates to a method ofproducing parvovirus capsids comprising the steps of:

i) infecting an insect cell with the recombinant baculovirus encodingthe major structural protein or co-infecting an insect cell with both ofthe above-described recombinant baculoviruses;

ii) culturing the cells under conditions such that the major structuralproteins are produced and self assemble to form the capsids; and

iii) isolating the capsids.

In yet a further embodiment, the present invention relates to a methodof producing a protein presenting capsid comprising the steps of:

i) coinfecting an insect cell with (a) a first recombinant baculovirusencoding a major structural parvovirus protein and (b) a secondrecombinant baculovirus encoding the nonunique region of a minorstructural parvovirus protein and a nonparvovirus protein;

ii) culturing the cells under conditions such that the expressedproteins self assemble to form the capsids; and

iii) isolating the capsids.

In another embodiment, the present invention relates to a proteinpresenting capsid comprising a major structural parvovirus protein and anonunique region of a minor structural parvovirus protein joined to anonparvovirus protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Human DHFR minigene DM14.

FIG. 2. Structure of the B19 capsid expression vector.

FIG. 3. Amplification of B19 capsid genes.

FIG. 4. Immunoblot of B19 capsid proteins in CHO and bone marrow cells.

FIG. 5. Immunofluorescence of a capsid-producing Chinese hamster ovary(CHO) cell-line: FIG. 2A—control CHO cells, and FIG. 2B transformed CHOcells.

FIG. 6. Sedimentation of B19 capsids.

FIG. 7. Electron micrograph of transformed CHO cells—demonstration ofintranuclear viral particles.

FIG. 8. Growth curves.

FIG. 9. Plasmid constructions containing the major (VP2) and minor (VP1)capsid genes of B19 parvovirus. (A) Diagram outlining relationship ofinserts derived from pYT103c, a nearly full-length molecular clone ofparvovirus B19, and the baculovirus vector pVL941; (B) synthesizedregions of DNA used to complete the gene sequences.

FIG. 10. Expression of B19 parvovirus proteins in insect cells infectedwith recombinant baculoviruses. (A) Immunofluorescence of Sf9 cellsinfected with pVP1/941 (a), pVP2/941 (b), and wild type baculovirus (c)after staining with convalescent phase antiserum to B19 parvovirus (x1500). (B) Immunoblot of lysates from cells infected with pVP1/941 (a),pVP2/941 (b) both pVP1/941 and pVP2/941 (c), and wild type baculovirusafter development with convalescent phase antiserum followed by¹²⁵I-labeled protein A. (C) Coomassie blue dye-stained polyacrylamidegel of Sf9 cell lysates after infection with recombinant baculoviruspVP1/941 (a), pVP2/941 (b), both pVP1/941 and pVP2/941 (c), and wildtype baculovirus (d).

FIG. 11. Electron micrographs of empty capsids. After infection witheither pVP1/941 plus pVP2/941 or pVP2/941, cell lysates were subjectedto equilibrium density gradient sedimentation and examined bytransmission electron microscopy after negative staining (x 171,000).

FIG. 12. Capture immunoassay comparing antigen derived from serum ofinfected patients with Sf9 c311 lysate after in vitro infection withpVP2/941.

FIG. 13. Shows supernormal amounts of VP1 were present in therecombinant capsids when the relative multiplicity of infection for VP1and VP2 baculoviruses was increased from 1:1 (7% VP1) to 100:1 (30%VP1). These results have two significant implications. First, becauseVP1 is immunogenic and probably contains the receptor binding site, acapsid enriched for VP1 compared to virion may be particularly effectiveas a vaccine reagent because it increases the amount of desirableantigen presented to the immune system. Second, the unique region of theVP1 plasmid could be replaced with other epitopes and other recombinantbaculoviruses generated. In this way, the basic capsid structure couldbe used to present multiple or different antigens to the host (that is,tetanus, gp120 of HIV) in the context a stable, highly immunogenicparticulate structure.

FIG. 14. Neutralization of B19 parvovirus infectivity for humanerythroid progenitors. Sera from six rabbits immunized with partiallypurified capsids composed of VP2 alone or VP2 and VP1 were tested fortheir ability to abrogate the toxic effect of B19 parvovirus in bonemarrow cultures.

FIG. 15. Schematic representation of a protein presenting capsid.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of producing parvovirusstructural proteins utilizing recombinant DNA techniques, to expressionvectors containing DNA sequences encoding the structural proteins, andto cells transformed with such recombinant molecules. The presentinvention also relates to recombinant baculoviruses encoding parvovirusstructural proteins and to insect cells infected with such recombinantviruses. The invention further relates to diagnostic assays utilizingthe recombinantly produced parvovirus protein products, or antibodies tosuch proteins. The invention also relates to a vaccine effective againstparvoviral infection comprising the recombinantly produced viral proteinproduct. The invention further relates to methods of treating diseasesamenable to genetic therapy, i.e., hemoglobinopathies and enzymedeficiency states, utilizing the recombinantly produced parvovirusprotein products, specifically parvoviral capsids, in celltransfections. The present invention further relates to methods ofproducing protein presenting capsid vehicles.

The present invention developed from Applicants' discovery that empty,and thus non-infectious, parvovirus capsids can be produced from themajor and minor parvovirus capsid protein species or from the majorparvovirus capsid protein species alone, without the non-structuralproteins. (The minor structural protein alone can not form a capsid.)The elimination of the noncapsid proteins allows for the production ofparvoviral particles, microscopically indistinguishable from infectiousparticles, which are incapable of killing the host cell.

In one embodiment, the present invention relates to a method ofproducing parvovirus structural proteins, for example, B19 structuralproteins, utilizing recombinant DNA techniques. Advantageously, thestructural proteins self assemble in the host cell (eucaryotic orprocaryotic) to form an empty, but intact, parvoviral capsid. Quantitiesof parvovirus capsids equal to or greater than those present in infectedbone marrow cells, can be produced by the method of the invention.

In a preferred embodiment, eucaryotic cells are transfected with arecombinant DNA molecule comprising an expression vector and the codingsequences of either the major capsid protein or the major and minorcapsid proteins of a parvovirus, under control of a promoter. Forselection, cells carrying a marker that alters the phenotype of the cellare used as the host. The recombinant DNA molecule containing thecapsid-encoding sequences is cotransfected with the sequence encodingthe marker gene (i.e., a gene encoding an enzyme deficient in theuntransfected cell). Transformants having the appropriate phenotype arereadily selected by growing the cells in a selective medium. (Cells canbe selected positively or negatively; negatively by the presence of agene conferring resistance in selective medium and positively by theexpression of a detectable marker allowing for identification andisolation of positive cells.) Such transformants are then screened,using known techniques, to determine which contain the capsid proteins.The capsid proteins are isolated in substantially pure form usingprotocols known in the art.

In a most preferred embodiment, CHO cells deficient in dihydrofolatereductase (DHFR) are cotransfected with: i) a recombinant DNA moleculecomprising an expression vector and a DNA sequence encoding the two B19parvovirus capsid proteins driven by the strong single B19 promoter, andii) a human DHFR minigene driven by the SV40 early promoter enhancerunit. Transformants bearing the DHFR+ phenotype are selected by growingthe cells in a medium lacking nucleosides; colonies are screened by RNANorthern analysis for expression of B19 genes. Coamplification of theintegrated B19 capsid-encoding sequence and the DHFR sequence can beaccomplished by treating the cells with increasing concentrations ofmethotrexate; coamplification results in detectable levels of proteinexpression.

Empty B19 parvovirus capsids are found in the nuclei and cytosol of theCHO cells transfected and cultivated as described above. Largequantities of capsids are not released into the culture supernatants.The expression of the empty capsids does not affect growth of the CHOcells.

In another embodiment, the present invention relates to a method ofproducing parvovirus capsids utilizing baculoviruses, for example,Autographa California nuclear polyhedrosis virus. Capsids composed ofthe major and the minor structural proteins and capsids composed of themajor structural protein alone are produced. The resulting capsids aresuitable reagents for human use and are easy to produce and purify.

In a preferred embodiment, recombinant baculoviruses are produced whichencode the major structural parvovirus protein, the minor structuralparvovirus protein, or a fusion protein. To form an empty, but intactparvovirus capsid containing the major and the minor structuralproteins, host insect cells (for example, Spodoptera frugiperda cells)are either infected with recombinant baculoviruses encoding bothstructural proteins or are coinfected with recombinant baculovirusesencoding the major protein species and recombinant baculovirusesencoding the minor protein species. Preferably, the cells are infectedwith viruses at a multiplicity of infections ranging from 10 to 50. Theinfected cells are cultured and the capsids self assemble. The assembledcapsids are isolated therefrom. Capsids composed of only the majorstructural protein are produced by infecting insect cells withrecombinant baculoviruses encoding the major protein.

That capsids, morphologically indistinguishable from natural emptycapsids, assemble from the major parvovirus structural protein, VP2,alone was unexpected. In computer modeling, VP1, the minor parvovirusstructural protein species, has been proposed as an internal proteinthat stabilized the capsid structure (Parvoviridae, In Animal VirusStructure ed. Nermut and Steven, Elsevier, N.Y. 325-334 (1987)). Thepresent data indicate instead that the minor structural protein is notrequired for capsid assembly; in fact, the minor structural proteinalone is incapable of forming a capsid structure. Several pieces of dataare consistent with the presence of VP1 on the surface of the capsidstructure. Only VP1-containing capsids allow production of neutralizingantibodies in rabbits. Individual human convalescent phase antisera andpooled immunoglobin recognize mainly VP1 rather than VP2, even thoughthe main acid sequence of VP2 is entirely contained within VP1; antiserathat recognize predominantly VP1 are equivalent to antisera thatrecognize VP2 in their ability to neutralize virus (J. Clin. Invest.84:1114-1123 (1989)). VP1's unique region of 228 amino acids contains atleast one neutralizing epitope by peptide mapping.

The minor structural protein appears to be more immunogenic than themajor protein. For example, most convalescent sera, that is sera frompeople who have been exposed to the virus previously, recognizepredominantly the minor structural protein. The minor structural proteinmay be the binding site for the receptor. Empty capsids having a higherminor structural protein to major structural protein ratio are usefulfor immunization as the number of neutralizing sites is increased.

In a preferred embodiment, capsids enriched with the minor structuralprotein are produced by altering the relative amounts of VP1 and VP2containing baculoviruses, so that the ratio of VP1 to VP2 is between10:1 to 100:1.

In another embodiment, the present invention relates to a safe andeffective method of producing antibodies against parvovirus capsids. Themethod comprises immunizing a mammal with the non-infectious, emptyparvoviral capsids described above comprising the major and the minorstructural proteins, using protocols known in the art, and isolating theantibodies produced. Monoclonal antibodies specific for the parvoviralcapsid can also be produced and isolated using known techniques. In apreferred embodiment, the antibodies, or useful binding fragmentsthereof, are specific for an epitope present on the B19 capsid.

In another embodiment, the present invention relates to a vaccineeffective against parvoviral infection. The vaccine includes the empty,non-infectious capsids described above (or an immunogenically effectiveportion thereof), purified so as to be essentially free of otherproteins (that is, so as to be safe for use as a vaccine). The capsidsare preferably, composed of both the minor and the major structuralproteins since neutralizing antibodies are elicited by capsidscontaining the minor structural protein and are not elicited by capsidscontaining only the major structural protein. In a preferred embodiment,the capsids are B19 capsids. Naturally VP1 accounts for 3-5% of theprotein in virion. VP1 also accounts for about 3-5% of the protein incapsids produced in host cells coinfected at a ratio of 1:1 withbaculoviruses encoding VP1and baculoviruses encoding VP2. When hostcells are coinfected at a ratio of between 10:1 and 100:1 withbaculoviruses encoding VP1and baculoviruses encoding VP2, the amount ofVP1is increased to 25-30% of the capsid proteins (see FIG. 13).

The invention also relates to diagnostic assays and kits based thereonfor detecting the presence in a biological sample of either parvoviralantigens or antibodies thereto. When parvoviral antigens are sought tobe detected, antibodies specific for same, produced as described above,can be used according to known protocols to effect antigen detection.When antibodies are sought to be detected, the above-described empty,non-infectious parvoviral capsids (or portions thereof recognized by theantibody), can be used as the antigen, in accordance with knowntechniques. Capsids containing the minor and the major structuralproteins as well as capsids containing only the major structural proteincan be used as the antigen. It is contemplated that immunodeficientindividuals incapable of producing antibodies against parvovirus can bedetected by challenging such individuals with the empty, non-infectiouscapsid containing the minor structural protein described above anddetermining whether antibody is produced in response to the challenge.

The diagnostic kits of the invention comprise the above-describedantibodies (or binding fragments) and/or capsid antigens and reagents,such as ancillary agents, for example, buffering agents. Wherenecessary, the kit can further include members of a signal-producingsystem, numerous examples of which are known in the art.

In another embodiment, the present invention relates to methods forpackaging and delivering genetic material to the genome of a cell. Themethod comprises encapsidating the genetic material sought totransferred into the empty, non-infectious parvoviral capsid describedabove containing the minor and the major structural proteins, andintroducing the capsid into a host cell under conditions such that, onceinside the cell, the genetic material is released from the capsid andexpressed. In a preferred embodiment, adenoassociated virus DNA is usedas that vector system. (See Lebkowski et al. Mol. Cell. Biol. 8:3988(1988) and McLaughlin at al. J. Virol. 62:1963 (1988)).

Genetic material suitable for use in such a method includes genesencoding proteins useful in the treatment of genetic defects, forexample, hemoglobinopathies and enzyme deficiency states. Host cellsinclude, for example, mammalian stem cells.

In another embodiment, the present invention relates to a method ofproducing a protein presenting capsid (see FIG. 14). Protein presentingcapsid can be made by substituting nonparvovirus proteins, such as,antigenic epitopes, ligands, enzymes or peptide sequences, for theunique region of the minor structural protein (e.g., VP1). (The uniqueregion of VP1 contains amino acids 1-226.) Recombinant baculovirusesencoding the modified minor structural protein can be produced usingmethods known in the art. The recombinant baculovirus can then be usedto coinfect an insect cell together with a recombinant baculovirusencoding the major structural protein to effect self-assembly of aparvovirus capsid having nonparvovirus proteins expressed on the surfacethereof. Such capsids can be used for example, to present antigenicepitopes for vaccination purposes.

Epitopes which can be substituted for the unique region of VP1include,for example, vaccine epitopes, such as diphtheria or pertussis epitopes.Further, capsids expressing multiple epitopes (for example, pertussisand B19 and diphtheria), can be generated using multiple recombinantminor structural protein genes. The use of such capsids in vaccineseliminates the use live vaccine and therefore related complications.

In addition, the unique region of the minor structural protein can bereplaced with a ligand for a cell surface receptor or an enzyme. Capsidsincluding ligand proteins can be targeted to certain cells. For example,capsids expressing a portion of a growth factor molecule would only bindto cells that had a receptor for that molecule. Capsids of the presentinvention can also be used to deliver enzymes to the circulation systemto treat diseases. As it is contemplated that different proteins can beexpressed on a single capsid surface, enzymes that attack, label ordestroy cells (for example, performs which poke holes in cells), can becombined with ligands that target the capsid to a cell to effectefficient cell killing or labeling. Such capsids can be used as generaldelivery system for proteins.

The protein presenting capsids of the present invention can also be usedin vitro as well as for therapeutic treatment. For example, the proteinpresenting capsids can be used in assays, such as immunoassays for thedetection of antibodies to various proteins.

The following non-limiting Examples describe the invention in moredetail.

EXAMPLE I Preparation of Recombinant DNA Molecules and Transfection ofCHO Cells

The DHFR minigene employed consisted of the entire encoding region ofthe DHFR gene and included the first intron; this construct was derivedby restriction enzyme digestion and ligation from that original DHFRminigene, DM-1 (Molec. Cell. Biol. 7:2830, 1987). The promoter-enhancerand polyadenylation signals were derived from the SV40 virus. Fortransfection, the DHFR minigene was cloned in pUC19 (see FIG. 1).

To prepare the B19 capsid expression vector, the nearly full-length B19genomic clone pYT103 c was digested with the enzymes EcoR1 and Aat andsubcloned into the standard vector pLTN-1. The nonstructural region wasdeleted by digestion with Xbal and Smal enzymes and recircularized (seeFIG. 2).

CHO cells were cotransfected with DNA from two plasmid constructs, onecontaining the DHFR minigene and the other containing the B19 capsidgenes. Transformants bearing the DHFR+ phenotype were selected bygrowing the cells in medium lacking nucleosides and colonies werescreened by RNA Northern analysis for expression of B19 genes.Coamplification of the integrated B19 capsid encoding sequence and theDHFR sequence was accomplished by treating the cells with increasingconcentrations of methotrexate. 3-11-5 is a cell line established asdescribed above which expresses the B19 capsid.

EXAMPLE II DNA and RNA Analysis

DNA was prepared by conventional phenol-chloroform extraction andproteinase K digestion and RNA by the conventional guanidinium sulfatemethod from 3-11-5 cells before and after culture in increasingconcentrations of methotrexate (final concentration=10 μM). DNA wasanalyzed by Southern and RNA by Northern hybridization using pYT103c, aB19 specific labeled DNA probe (Science 233:883 (1986)). The migrationon agarose gel electrophoresis of the B19 DNA from 3-11-5 cells isconsistent with the size of the transfected DNA insert and that of theRNA with the transcripts expected from the right side of the virusgenome (J. Virol. 61:2395 (1987)) (see FIG. 3).

EXAMPLE III Comparison of B19 Capsid Accumulation by Immunoblot

3-11-5 cells were compared to normal or erythroid bone marrow cellsinoculated with virus and harvested at 48 hours (the peak of virusproduction; Blood 70:384 (1987)). Capsid protein was detected by Westernblot using convalescent phase antiserum containing high titer anti-B19capsid protein IgG (J. Virol. 61:2627 (1987)) (see FIG. 4). The amountof 58 kd and 83 kd protein in 3-11-5 cells was intermediate between thatharvested from cultures of normal and erythroid bone marrow. Fromcomparison to known standard plasma preparations, it has been estimatedthat each 3-11-5 cell contains between 1000-20000 capsids.

EXAMPLE IV Immunofluorescence

3-11-5 and control CHO cells ware fixed with acetone and stained withhuman convalescent phase serum containing anti-B19 capsid antibodiesfollowed by fluorescein-conjugated anti-human IgG (J. Clin. Invest.74:2024 (1984)). All 3-11-5 cells show a pattern of strong and specificimmunofluorescence in both cytoplasm and nuclei (see FIG. 5).

EXAMPLE V Sedimentation Analysis of Capsids from 3-11-S Cells

Capsids from CHO 3-11-5 cells were compared to viral particles fromhuman bone marrow culture Blood 70:385 (1987)). Proteins were labeled byexposure of cultures to 35S-methionine, the cells were lysed, and theparticulate fraction obtained by centrifugation over a 40% sucrosecushion (J. Virol. 61:2627 (1987)). After suspension of the particulatefraction in a small volume of buffer, radioactively labeled capsids orvirions were applied to sucrose (J. Clin. Invest. 73:224 (1984)) orcesium chloride (Science 233:883 (1986)) gradients (see FIG. 6). Onsucrose gradient sedimentation, empty capsids were clearly distinguishedfrom intact virions, and isopycnic sedimentation in cesium showed adensity consistent with empty capsids.

EXAMPLE VI Electron Microscopy of 3-11-5 Cells

Cells were fixed and prepared for transmission EM as described (J. Clin.Invest. 74:2024 (1984)). Characteristic clusters of 20 nm particles wereobserved in the nuclei of 3-11-5 cells only (see FIG. 7).

EXAMPLE VII Growth Curves of 3-11-5 Cells Compared to Other CHO Cells

Cells were serially harvested from microtiter wells and manuallycounted. Empty capsid production does not adversely affect cellproliferation of 3-11-5 (see FIG. 8).

EXAMPLE VIII Preparation of Recombinant Baculoviruses, Transfection ofSf9 Cells and Expression of Capsids

Cell culture and virus stocks were prepared as follows. Recombinantplasmid was used to generate recombinant baculoviruses. AutographaCalifornia nuclear polyhedrosis virus (AcMNPV) and recombinantpolyhedrosis viruses were grown in monolayers of Sf9 cells. The Sf9 cellline (American Type Culture Collection, Rockville MD), which is derivedfrom Spodoptera frugiperda (fall army worm) ovary, was maintained inGrace's insect tissue culture medium containing 10% heat inactivatedfetal bovine serum, 2.5 μg/ml fungizone, 50 μg/ml gentamicin, 3.33 mg/mllactalbumin hydrolysate, and 3.33 mg/ml yeastolate (provided complete byGibco BRL Life Technologies, Gaithersburg Md.) at 100% room air, 95%humidity, at 27° C.

Recombinant plasmids and recombinant baculoviruses were constructed asfollows. Two plasmids were constructed, one containing the full lengthmajor capsid protein gene (VP2), the other the full length minor capsidprotein gene (VP1). To construct plasmid pVP1/941, a cDNA encoding theVP1gene was excised from pYT103c, a nearly full length molecular cloneof B19 parvovirus (Cotmore et al. Science 226:1161 (1984); Ozawa et al.J. Virol. 62:2884 )1988)), by digestion with the restriction enzymesHind III (which cuts at map unit 45) and EcoRI (which cuts at map unit95) followed by treatment with mung bean nuclease to complement singlestranded ends.

The resultant DNA fragment was inserted into the BamHI site (made bluntended with the Klenow fragment of DNA polymerase) of the baculovirustransfer vector pVL941, a vector derived by deletion of the polyhedringene of AcMNPV followed by cloning into the pUC8 plasmid (Summers et al.Tex. Agric. Exp. Stn. 1555 (1987)). Construction of pVP2/941 wasperformed by the insertion of a PstI-EcoRI digestion fragment of pYT103c(map units 58-95; the EcoRI site was blunt-ended) and a synthetic DNAfragment of 20 nucleotides corresponding to the SstI-PstI region (againwith the SstI site blunt-ended) into the BamHI site of pVL941 (FIG. 9).

Recombinant plasmids were used to generate recombinant baculoviruses.Eight μg of each of the recombinant plasmids was cotransfected into Sf9cells with 2 μg of wild type AcMNPV, using calcium phosphate-mediatedprecipitation. Six days after transfection, progeny virus was harvestedand replaqued onto fresh Sf9 cells. Recombinant viruses were recognizedvisually by the absence of occlusion bodies in the nucleus of cells (theocclusion-positive phenotype is the result of synthesis of largequantities of the polyhedrin protein). Recombinant viruses weresubjected to three cycles of plaque purification before large scalevirus stocks were prepared.

For analysis of protein expression and capsid structure, Sf9 cells wereinfected with recombinant viruses at multiplicity of infections (m.o.i.)ranging from 10 to 50. Cells were harvested and examined for expressionof VP1and VP2 at variable times after infection; four dayspost-infection was judged optimal for recombinant protein expression.Cytocentrifuge preparations (approximately 1×10₃ cells/slide well) ofrecombinant (VP1-VP2 baculovirus) or wild type virus-infected cells werefixed in acetone at −20° C. for 30 seconds, washed twice in phosphatebuffered saline (PBS) containing 0.5% bovine serum albumin, and blotteddry. Cells were stained with convalescent phase human anti-B19parvovirus antiserum (diluted 1:20), followed by application offluorescein isothiocyanate-conjugated goat antihuman IgG (diluted 1:50:Kierkegaard and Perry, Gaithersburg, Md.).

All cells stained specifically with the human convalescent phase humanantiserum, with bright fluorescence observed over cytoplasm and nucleiof fixed cells (FIG. 10); the fluorescent signal was maximal 3-4 daysafter infection and had faded after one week of culture, at which timemost of the cells were no longer viable.

For analysis of proteins by gel electrophoresis, lysates from 4 day oldcultures were prepared by heat disruption at 100° C. for 3 minutes in100 μl of Laemmli sample buffer(Nature 227:680-685 (1970)). Aliquots ofeach sample were applied to 8% polyacrylamide gels (10 μl/lane) in thepresence of sodium dodecyl sulfate as described by Laemmli. Proteinswere directly visualized by staining with visualized by staining with0.25% Coomassie brilliant blue dye. For immunoblotting, proteins weretransferred by eletroblotting onto nitrocellulose membranes (HoefferScientific, San Francisco Calif.). Specific proteins were detected bysequential application of convalescent phase human antiserum (dilutedI:300) and ¹²⁵I-labeled protein A (Amersham, Arlington Heights Ill.) bythe BLOTTO method (GeneAnal. Tech. 1:3-8 (1984)).

Bands of the appropriate molecular weight were detected after infectionwith the VP1-baculovirus (FIG. 10B, lane a), VP2-baculovirus (FIG. 10B,lane b), or after coinfection with both recombinant viruses (FIG. 10B,lane c). Large enough quantities of parvovirus structural proteins wereproduced to be visible after dye staining of polyacrylamide gels oflysates (FIG. 10C); parvovirus protein was estimated by densitometry toconstitute 2-3% of total cell protein present.

Capsids were examined by electron microscopy after equilibrium densitygradient sedimentation. Sf9 cells were harvested 4 days afterinoculation with recombinant baculoviruses (VP1alone, VP2 alone, orVP1plus VP2). Lysates were centrifuged at 100,000×g over 40% (wt/vol)sucrose in Hank's balanced salt solution. Precipitates were mixed withCsCl in 50 mM Tris-HCl, pH 8.7, 5 mM EDTA, and 0.1% sarcosyl at aninitial density of 1.31 gr/ml, centrifuged at 100,00×g in an SW41 rotorfor 35 hrs at 18° C. Transmission electron microscopy was performedafter three such banding procedures.

Banding of parvovirus proteins (determined by immunoblot andimmunoprecipitation) was detected at 1.31 gr/ml, the appropriate densityfor empty capsids, for cells infected with VP1-baculovirus and cellscoinfected with VP2 and VP1-baculoviruses. No parvovirus protein wasdetected in cell lysates from VP1-baculovirus infected cells.

Direct electron microscopy was done on pellets after ultracentrifugationof 50 μl of the sample in 3.5 ml Dulbecco A PBS. Immune electronmicroscopy was performed by incubating 50 μl of human serum containingIgG antibody to B19 parvovirus for 45 minutes at 20° C. prior todilution in PBS and ultracentrifugation. Pellets after centrifugationwere resuspended in 50 μl of distilled water and negatively stainedusing 3% phosphotungstic acid, pH 6.5. Grids were examined at60,000×magnification in Jeol 1200EX electron microscope. Magnificationswere calibrated with catase.

Immune electron microscopy showed typical empty parvovirus capsids,aggregated by the B19 antibody, in samples from cultures coinfected withVP1and VP2-containing baculoviruses and also in cultures after infectiononly with VP2-baculoviruses (FIG. 11). No virus particles were seen inlysates of cells infected with VP1-containing baculovirus alone. Directelectron microscopy of harvests from cultures coinfected with VP1andVP2-containing baculoviruses and from cultures infected withVP2-containing virus only revealed numerous typical parvovirus-likeparticles that were not coated with antibody. A minority of theparticles were electron dense, the majority were less dense, and someparticles had intermediate density. Particles tended to clustertogether.

The capture immunoassay was adapted from a previously published Elisaimmunoassay procedure(J. Clin. Microbiol. 24:522-526 (1986)). Captureantibody, either goat anti-human IgG or IgM antibodies (Tago, BurlingameCalif.) was added to 96 2311-microtiter plates (Immunolon, Dynatech,Alexandria Va.) and incubated for 1 hour at 37° C.; the plates werewashed and human serum specimens (diluted 1:100) were added for 1.5hours at 37° C. After washing, positive and control antigens were addedovernight at room temperature; antigens included pooled sera fromviremic human specimens and baculovirus-expressed antigen. Furtherwashing of the plates to remove antigen was followed by addition ofbiotinylated monoclonal antibody (MAb 521-5d, diluted 1:2000) for 1 hourat 37° C., another wash step, and addition of peroxidase-conjugatedstreptavidin (plc, Amersham International, United Kingdom) for 10minutes at room temperature. Substrate for the enzyme (0.1 mg/ml of3,3′5,5′-tetramethyl-benzidine and 0.005% H₂O₂ in dimethyl sulfoxide andacetate-citrate buffer, pH 5.5) was added to the plates after furtherwashing for 15 minutes at room temperature; the reaction was stoppedwith 2 M H₂SO₄ and the absorbance at A₄₅₀ determined. Capture antibodywas diluted in 0.01 M carbonate buffer, pH 9.6; other reagents werediluted in phosphate buffered saline (pH 7.2) with 0.5% gelatin and0.15% Tween-20; plates were washed with phosphate buffered saline, 0.15%Tween-20.

Each serum specimen was tested in duplicate against baculovirus antigenand negative control antigen at 1:2000 dilution and against a humanserum pool of viremic blood at a dilution of 1:200. A serum specimen wasconsidered positive in the baculovirus IgG immunoassay if the P-Nwas >0.35 and the P/N ration was >2.0, in the baculovirus IgMimmunoassays if P-N was >3.0 and P/N was >2.0, and in the human serumIgG and IgM immunoassays if P-N was >3.0 and P/N was >2.5. P is the meanabsorbance for the serum specimen reacted against the B19 viral antigenless mean absorbance due to nonspecific binding of B19 viral antigen(negative serum or diluent reacted against positive antigen minusnegative serum reacted against control antigen). N is the meanabsorbance for the same serum specimen reacted against the respectivenegative control antigen. These values of P-N and P/N are ≧3 standarddeviations above the mean values for specimens previously determined tobe antibody-negative.

For the IgG assay, 23 specimens were negative in both immunoassays, 45were positive in both assays, and none was discordant. For the IgMassay, 25 specimens were negative in both assays, and none wasdiscordant. The assays based on the two different sources of antigenalso gave comparable qualitative results (FIG. 12). The correlationcoefficients for P-N absorbance values for serum antigen versusbaculovirus antigen wa 0.95 for the IgG immunoassays and 0.91 for theIgM assay.

For the production of antisera, rabbits were immunized with partiallypurified empty capsids obtained after coinfection of insect cells witheither VP1and VP2-containing baculovirus or with only VP2-containingbaculovirus. After lysis, capsids were subjected to sedimentation oversucrose and in cesium chloride, as described above. Animals wereinoculated with either 20 or 200 μg of capsid protein by subcutaneousinjection, initially in complete Freund's adjuvant and with boosterinjections in incomplete Freund's adjuvant at 2-4 week intervals. Rabbitsera were analyzed by immunoblot and in neutralization assays.

To determine neutralizing activity, sera were heated to 56° C. for 30minutes to destroy complement activity and incubated at varyingconcentrations with quantities of B19 parvovirus known to inhibiterythropoiesis in vitro. The inhibitory activity of virus treated withantiserum was compared to virus alone in conventional assays of lateerythroid progenitors (CFU-E), cultured in 0.8% methylcellulosecontaining 30% fetal calf serum, 1% bovine serum albumin, 10⁻³beta-mercaptoethanol, and 1 μ/ml recombinant erythropoietin (Amgen,Thousand Oaks Calif.) at 37° C., 95% humidity for 6-7 days. Controlexperiments included assay of preimmune rabbit sera and similarlydiluted normal human sera that had been obtained from patients in theconvalescent phase of parvovirus infection; these sera containedantibody to B19 parvovirus, as determined in the capture immunoassay.

None of the animals inoculated with low doses of antigen (20μg/injection) produced neutralizing antisera. However, in {fraction(3/3)} animals immunized with larger quantities of empty capsids (200μg/injection), composed of both VP1and VP2, obtained after coinfectionof insect cells with the two individual recombinant viruses,neutralizing antisera was produced. The titers of neutralizing activityin two animals were comparable to those observed in convalescent phasehuman sera. (FIG. 14 shows the production of neutralizing antisera inresponse to capsid containing both VP1and VP2).

In contrast, none of three sera from animals immunized withVP2-containing capsids produced neutralizing antibody. Ouchterlonyanalysis was used to determine if precipitating antibodies were made bythese animals, using empty capsids made in mammalian cells or VP2-onlycapsids produced in baculovirus as antigens: sera from the animals whichhad produced neutralizing antibodies after immunization with VP1and VP2also contained precipitating antibodies, and sera from the animalsimmunized with VP2-capsule also demonstrated precipitating antibodies.

The foregoing invention has been described in some detail by way ofexamples for purposes of clarity and understanding. It will be obviousto those skilled in the art from a reading of the disclosure thatsite-directed mutagenesis can be used to alter the amino acid sequenceof the above described capsids and thereby alter the tissue specificityof the virus. Furthermore, it will be clear that the DHFR-deficient CHOcells can be used to study the effect of nonstructural parvoviralproteins on cell replication. It will also be apparent that variouscombinations in form and detail can be made without departing from thescope of the invention.

The entire contents of all published articles cited herein are herebyincorporated herein by reference.

What is claimed is:
 1. A method of producing antibodies to B19parvovirus capsids comprising: immunizing a mammal with a non-infectiousempty B19 parvovirus capsid comprising a full length VP2; and isolatingthe antibodies produced in the mammal.
 2. The method of claim 1 whereinthe non-infectious empty B19 parvovirus capsid further comprises VP1.